Notes

Abstract:

Endosperm damage was studied in Monteverde, Costa Rica to determine its frequency, extent, and effect on seed germination and early seedling growth. Endosperm damage was found to occur in 72% of seeds from Chione sylvicola (Rubiaceae) and 94% of seeds from Quercus costaricensis (Fagaceae). The extent of endosperm damage in Q. costaricensis was not related to the root and shoot weight of germinated seeds. Experimental endosperm removal was carried out in Persea americana and Cinnamomum paratriplinerve (both Lauraceae) seeds. The three week time period of the study was inadequate to yield significant C. paratriplinerve germination, but seeds with intact endosperm were much less likely to suffer fungal infection. Endosperm removal did not inhibit germination in P. americana seeds, even when 80% of the original seed weight had been removed. After 23 days in soil, most seeds were just beginning to produce roots and shoots, the majority of which were still enclosed in endosperm. At this time, P. americana seeds with a higher percent of endosperm removed had significantly heavier roots and shoots, suggesting a reduced or even inhibitory role of endosperm in initial seed germination.

even a few extra millimeter s of shoot or root length can make a great difference competitively for a seedling (Leishman et al. 2000). As larger seeds produce larger seedlings, larger seed seedlings are able to grow up out of greater depths of soil, where the seed can receive more mo isture. Seedlings of larger seeds also emerge from leaf litter more successfully. Larger seeds tend to have a higher percentage of their mass stored as reserve that is gradually deployed to permanent structures. More reserve remains in the seed uncommitted available for the seed to draw upon in case of undesirable growing conditions or seedling disturbance. Slower germination and growth can put larger seeds at a competitive disadvantage, since less tissue is available for photosynthesis. However, studies h ave concluded that larger seeds have an advantage in times of drought, intense shade, and mineral nutrient deficiency (Leishman et al. 2000). Dispersal modes are also associated with seed size (Harper et al. 1970, Primack 1987). Larger seeded species are t ypical of the mammal dispersal syndrome, (Foster and Janson 1985) but little evidence exists conclusively linking seed size with distance of dispersal (Leishman et al. 2000). Loss of endosperm to predation or parasitism is widespread in tropical forests d ue to the length spent on the forest floor (Calvo Irabin and Islas Luna 1999; Dalling, Harms, and Aizpra 1997). Tropical canopy trees, seeds and seedlings commonly suffer severe damage from insects and fungi (Clark and Clark 1991). The role of seed herbi vores, or granivores, in selecting for seed size in the tropics has been reported in multiple studies. Harms and Dalling (1997) examined 13 dicotyledon tree species from Barro Colorado Island (BCI), with seeds ranging in weight from 0.2 to 107.6 grams. Aft er each seed produced its first pair of fully expanded leaves, they clipped the above ground shoot and found that only seeds heavier than five grams were capable of resprouting and producing fully functional seedlings. These results agree with a similar st udy for seed damage to Macuna andreana (Fabaceae), a tropical liana with seeds weighing five to ten grams (Janzen 1976). Further, Ichie et al. (2001) show that germination and seedling growth are accompanied by depleted starch and lipid reserves in Dryobal anops lanceolata (Dipterocarpaceae). Once depleted, seedling growth ceases, suggesting that seedling size is a direct result of seed reserves. Dalling, Harms, and Aizpra (1997) studied the germination, initial seedling production, and seed and seedling da mage tolerance of Prioria copaifera (Fabaceae), the largest seeded tree species on Barro Colorado Island, Panam. They found that experimental endosperm removal of up to 60% of seed mass did not effect the proportion of seeds germinated, but decreased shoo t mass. This supports the portrayal of seed endosperm as a determining factor in seedling growth rather than germination. Janzen (1976) demonstrated that removal of only 1%, 5%, and 10% of M. andreana seed weight by drilling holes in the endosperm to mimic insect damage severely reduced seed fitness in the face of artificial granivory. Less seed reserves remained to be utilized to compensate for the herbivore damage. In the present study I first aimed to confirm the commonness of endosperm damage in the Cl oud Forest by examining the frequency of granivory in Chione sylvicola (Rubiaceae), a small seeded member of the coffee family whose seeds are commonly seen along Cloud Forest trails. I next examined the impact of natural and simulated granivory on germina tion and initial seedling growth for two other species of Cloud Forest trees, Quercus costaricensis (Fabaceae) and Cinnamomum paratriplinerve (Lauraceae), and along with the commercial avocado, Persea americana (Lauraceae). Specifically, I analyze the role of endosperm in seed germination ability and early seedling size. I note the degree of fungal infection in seeds with removed endosperm, and quantify the amount of endosperm missing and development of root and shoot from the two

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Cloud Forest species. Furt her, I test for whether root and shoot masses are enhanced by soil nutrients or are accumulated from endosperm stores alone by growing half of the avocado seeds in water rather than soil. MATERIALS AND METHODS Field Observations In mid November 2005 C. sylvicola seeds along a forest trail near the Monteverde Biological Station were examined. The first 100 red C. sylvicola. seeds lying exposed on the trail were inspected for granivore damage. These seeds, which were typically around 20 grams in weight, w ere categorized as damaged if they exhibited exit holes or had portions of the seed removed. While counting the 100 seeds a tally mark was recorded for each counted seed qualifying as damaged in order to determine the percentage of seeds preyed upon. Durin g the same week 100 Q. costaricensis (Fagaceae) seeds (mean weight = 2.26 g, sd = 0.68 g) were collected from the same trail. Q. costaricensis drops its seeds, or acorns, synchronously so all the seeds were of relatively the same age (Burger 1975). Alm ost all seeds were in the early stages of germinating, with a small leafless shoot only a few centimeters in diameter emerging from the center of the seed. To accurately measure seed size only seeds with both halves remaining were gathered. After the seeds were gathered any pieces of shell or debris attached to the seeds were removed and the root and shoots were clipped off. The number of insect exit holes was counted for each seed, and the percent of endosperm removed from the seed was estimated. Each pair of seed halves was weighed, along with their respective roots and shoots. Original seed weights were calculated using the measured seed weights and respective estimates of percent endosperm removed. Endosperm removal experiments 100 P. americana seeds w ere removed from their fruits. In order for this experiment to yield data, the seeds (at least the ones without endosperm removed) needed to be capable of germinating within the short time span allotted for this study. Out of this concern the seed coat was removed from each seed, as well as a sliver of tissue from both ends, as this has been shown to hasten germination (Eggers 1942). Each seed was weighed after this removal. To determine whether soil nutrients as well as endosperm impacted seedling growth, 50 of the P. americana seeds were grown in water and the remaining 50 were designated for soil treatment. Seeds were equally divided between manipulations of 0%, 20%, 40%, 60% and 80% endosperm removal by weight (Table 1). A knife was used to cut away tiss ue from the seed end distal to the embryo, leaving the actual embryo untouched. The water treatment seeds were each supported within the rim of a water filled plastic cup by three toothpicks, assuring that at least the embryo contain bottom two centimeters of the seed was submerged. The soil treatment seeds were planted in large germination trays with .5 1 cm of seed left exposed above the soil, which was a two to one mixture of forest soil and sand. Both treatments were placed indoors along a large westwar d facing window. The soil in the germination trays was kept moist and the water cups were kept at a constant level of water.

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TABLE 1. Mean weights of P. americana seeds in (a) the water treatment set and (b) the soil treatment set. (a) (b) % of endosperm removed Mean initial weight (g) Mean after cut weight (g) % of endosperm removed Mean initial weight (g) Mean after cut weight (g) 0 30.895 5.616 30.895 5.616 0 25.012 6.171 25.012 6.171 20 28.650 6.114 23.032 6.270 20 27.538 5.296 21.930 4.056 40 30.075 4.132 18.014 2.433 40 29.902 6.257 17.922 3.776 60 29.826 6.205 12.094 2.583 60 29.331 9.258 11.733 3.642 80 26.009 5.340 5.259 1.053 80 26.449 6.475 5.423 1.351 Fifty intact C. paratriplinerve seeds with their seed coats intact were weighed and endosperm was removed from them in the same mann er as with the P. americana seeds. Ten seeds were left completely intact, and 20%, 40%, 60%, and 80% endosperm removal were each performed on ten seeds (Table 2). All the C. paratriplinerve seeds were then planted in germination trays .5 cm deep in two:on e forest soil and sand mixture, and placed in the same window as the P. americana seeds. TABLE 2. Mean weights of C. paratriplinerve seeds planted in soil. % of endosperm removed Mean initial seed weight (g) Mean after cut seed weight (g) 0 0.493 0.077 0.494 0.077 20 0.493 0.060 0.395 0.049 40 0.503 0.057 0.300 0.032 60 0.478 0.077 0.190 0.029 80 0.442 0.067 0.087 0.012 All P. americana seeds were removed from their treatments 23 days following planting. The seeds were cut o pen and the sprouting roots and shoots, none of which had grown longer than a few centimeters, were then removed and weighed separately. Bivariate regression analyses were performed to test for correlations between the percent of endosperm removed and the resulting root and shoot weight. The C. paratriplinerve seeds were removed after 22 days in soil and all seeds were examined for shoots or roots. If the seed had germinated the shoot and root were weighed together. Each seed was also inspected for fungal i nfection. RESULTS Field Observations Endosperm damage is common. Out of the 100 C. sylvicola seeds observed, 72 seeds exhibited herbivore damage. This damage was characterized by exit holes or partially missing endosperm. In damaged seeds, the soft ti ssue on the outside of the seed either had chunks taken out of it, showed small dark round bruises around tiny insect exit holes, was split laterally, or showed any combination of these signs of granivory. Seed damage ranged in severity from only one or tw o insect exit holes to the removal of over half of the endosperm. Quercus costaricensis seeds suffer an even higher frequency of damage than C. sylvicola Furthermore, damage in this species is cause by insects both boring holes into the seed endosperm and consuming its contents. Some of the seeds still contained insect larvae within

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them when examined in the lab. Only six of the 100 Q. costaricensis seeds gathered show no signs of endosperm damage. Seeds had an average of 11 13% of their endosperm estima ted to be missing (Figure 1a). Ten of the seeds had so little intact tissue remaining that it was impossible to count the number of insect exit holes in the seed. These seeds were excluded from exit hole analyses. With these seeds excluded the number of ex it holes in a seed averaged 28 17 (Figure 1b). FIGURE 1. Q. costaricensis seeds with (a) estimated percent of seed endosperm removed (n = 100) and (b) number of exit holes (n = 90). Insects do not prey preferentially upon larger Q. costaricensis seeds. The number of exit holes in a Q. costaricensis seed did not relate to either its measured weight (r 2 = 0.006, P = 0.486, n = 90; Figure 2a) nor its calculated original weight (r 2 = 0.026, P = 0.130, n = 90; Figure 2b). Since insect damage did not vary significantly by seed size, seeds of smaller weight on average suffered a greater proportion of endosperm loss. FIGURE 2. Significant regres sions relating the percentage of endosperm removed from Q. costaricensis seeds to (a) the measured seed weight (r 2 = 0.21, P < 0.0001, n = 100) and to (b) the calculated estimated original seed weight (r 2 = 0.086, P = .0032, n = 100). Insects damaging Q. costaricensis endosperm do not regularly damage the seed embryo or seedling. The shoot and root growth of the germinated seeds was not found to be significantly

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hindered by any amount of endosperm damage. The number of exit holes in a seed neither correlat ed to its root and shoot weight (r 2 = 0.0004, P = 0.845, n = 90; Figure 3a), nor to its root and shoot weight as a percentage of its calculated original weight (r 2 = 0.0006, P = 0.813, n = 90; Figure 3b). FIGURE 3. There was no significant correlation between either the number of insect holes removed from a Q. costaricensis seed and (a) the weight of its respective root and shoot nor (b) the weight of the root and shoot relative to the ori ginal seed weight. Endosperm removal experiments The large avocado seeds did not have inhibited germination even when 80% of their endosperm had been removed. Twenty three days after planting, 97% of the seeds had germinated. This germination was so subt le, however, that only about a quarter of the shoots had grown taller the top of the seed (mean root and shoot weight of water treatment seeds = 0.170 g, sd = 0.092 g; mean root and shoot weight of soil treatment seeds = 0.142 g, sd = 0.111 g). Most shoots and roots combined were only a centimeter longer than the embryo, and none had began to visibly deplete the endosperm reserves. Upon the extraction of seedlings from soil treatment seeds it was discovered that the 80% of the endosperm cut from one seed ha d been inadvertently removed from the wrong end, so that the seed no longer contained an embryo. This seed was excluded from analysis. By examining the contents of each seed when removing seedlings it was confirmed that this error did not occur in any of t he other 99 P. americana seeds used in the study. Seeds grown in soil differed from those grown in water in that removal of their endosperm increased their root and shoot weights. The water treatment seeds showed no significant correlation between either t he percent of endosperm removed and the respective root, shoot, root and shoot combined weight, or root and shoot combined weight as a percentage of the original weight of each seed. The P. Americana seeds grown in soil, however, showed a strong positive l inear relation was found for each of these relationships except the percent of endosperm removed versus root weight (Fig. 4).

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FIGURE 4. The effect of the percent of endosperm removed on (a) shoot weight (r 2 = 0.131, P = 0.011, n = 49), (b) combined root and shoot weight (r 2 = 0.104, P = 0.024, n = 49), and (c) combined root and shoot weight as a percentage of the initial seed weight (r 2 = 0.084, P = 0.044, n = 49) of P. america na seeds grown in soil. Only one C. paratriplinerve seed had germinated by the end of the growing experiment. This was insufficient data to draw conclusions on the effect of endosperm removal on germination growth. However, a large number of the seeds ha d developed a fungal infection. The fungus infested endosperm swelled up above the edges of the seed coat where the seed had been cut, and the endosperm was white and liquefied. All infected seeds were dissected to inspect for germination, and none of thei r embryos showed any sign of growth; some were so degraded they were barely distinguishable from the rest of the dissolving tissue. Intact seeds showed no infection, but all other categories, even those with as little as 20% removed, had complete or nearly complete infestation (Figure 5). A Chi squared sample test showed that that the number of seeds infected with the fungus varied significantly between the percent endosperm removed groups (X 2 = 9.85, df = 4, p < 0.05;). FIGURE 5 Cinnamomum paratriplinerve seeds with removed endosperm were more susceptible to fungal infection, while those still intact were untouched by the fungus. DISCUSSION Large seeds may be adaptive for many reasons. They are better equipped to respond to abiotic conditions important for tropical trees: shade, seasonal rain, depleted soils (Leishman et al. 2000), as well as the biotic conditions that are stronger in Tropical forests: competition,

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predation, and parasitism. Further, these conditions are not necessarily mutually exclusive. The current study explores the importance of granivory to germination and subsequent immediate seedling growth. Seed e ndosperm damage is common in lowland tropical forests (Calvo Irabin and Islas Luna 1999, Clark and Clark 1991, Dalling et al. 1997). Cloud Forest seeds seem to suffer the same high rates of endosperm loss as seeds from lower sites. Of the two seeds studie d in the field, both showed high rates of endosperm damage. Predation of C. sylvicola seeds was not directly witnessed so its specific granivore is unknown, but insect larvae still remaining in the Q. costaricensis allowed the identification of their damag er. Larger oak seeds suffered less damage proportionately than smaller oak seeds, as insects seemed to attack both large and small seeds with equal frequency. Larger seeds may also escape fungal infections longer, though here fungal infection resulted just from any endosperm removal. Seed size had no effect on the extent of its insect infestation nor did insect damage reduce root and shoot size. Insect infestation in acorns occurs most frequently on the basal end of the seed, opposite from the embryo, possi bly explaining why germination frequencies are not affected by endosperm removal (Steele et al. 1993). Germination experiments showed that early seedling development is not impacted by endosperm removal, even up to 80% removal. This initially seems to con flict with the P. copaifera study by Dalling, Harms, and Aizpra (1997). However, that study was conducted over a 38 week growing time, after which all the germinated seedlings had a fully developed pair of leaves. Seedlings from avocadoes were smaller tha n those observed in the oak seeds, and much smaller than those from the P. copaifera study, so it is likely due to their small size that only 20% of endosperm remaining was sufficient for their early growth. This successful germination after endosperm dama ge coincides with the initial results of a study on Acacia albida (Mimosaceae) seeds by Hauser (1994). Seeds showing bruchid beetle exit holes initially had a high germination rates. A few months later, though, they all had died. Other studies (Dalling et al. 1997, Ichie et al. 2001) suggest that later seedling development would expose the negative impact of endosperm removal on seedling size, however. It is predicted that though the experimentally endosperm deficient P. Americana seeds had greater initial germination growth, had the experiment been allowed to run longer their roots and shoots would have eventually been surpassed in weight by those of seeds containing more endosperm. This could put plants with more endosperm removed at a competitive disadva ntage, as taller seedlings and saplings are Finally, soil seedlings actually grew larger with more endosperm removed. It is likely that this was due to their earlier germination. Apparently the greater a mount of endosperm remaining on the more intact seeds inhibited their germination. The function of endosperm is to fuel growth of a seedling until it is capable of gathering energy from photosynthesis (Ichie et al. 2001); the mechanism of germination may b e more significantly fueled by an interaction between the seed embryo and the soil rather than the use of energy reserves in the endosperm. contact with soil compounds and thus may have hastened germination in soil treatment seeds with more endosperm removed. Perhaps avocado seeds grown in water did not show a similar trend in seedling size due to the their lack of soil substrate whose proximity could have given seeds wi th less endosperm around their embryo an advantage. Earlier germination among endosperm deprived soil treatment seeds may also explain why gap plants have smaller seeds, if fast germination time is more important for them than ultimate seedling size. Large seeds may